Two variant forms of human liver mitochondrial aldehyde dehydrogenase (ALDH) have been identified which differ from each other by just one amino acid substitution. ALDH 2E, which has Glu at position 487, is found in most individuals. It has low Km for acetaldehyde and is responsible most of the acetaldehyde oxidation in liver. ALDH 2K, which has Lys at position 487, is found in about 50% of Japanese and Chinese. It exhibits little or no acetaldehyde oxidizing activity. Since the enzymatic basis for this loss in activity of ALDH 2K is not known, we propose in this pilot project to purify ALDH 2K to homogeneity and compare its catalytic and structural properties to those of the active ALDH 2E isoenzyme. This is of interest, since there is little information regarding the roles of specific amino acids in the catalytic mechanism of ALDH. The dehydrogenase and esterase activities with different substrates and coenzyme binding properties will be examined. Chemical modification studies will be performed to see whether alkylation of Cys residues in both isoenzymes alters catalytic activity or coenzyme binding. We will examine the thermal stability of the native isoenzymes. We will also develop methods for identifying livers from individuals who are homozygous or heterozygous for the Aldh2K allele. Experiments will be performed to determine whether heterotetramers of ALDH 2E and ALDH 2K subunits are formed.